Adjuvant chemotherapy for anaplastic gliomas

ABSTRACT

The present invention involves the use of 2,4-disulfonyl phenyl tert-butyl nitrone (2,4-ds-PBN) in the treatment and prevention of gliomas. The 2,4-ds-PBN may be used alone or combined with other traditional chemo- and radiotherapies and surgery, to treat or prevent glioma occurrence, recurrence, spread, growth, metastasis, or vascularization.

This application is a continuation application of U.S. patentapplication Ser. No. 12/552,792, filed Sep. 2, 2009, now U.S. Pat. No.8,633,249, issued Jan. 21, 2014, which claims benefit of priority toU.S. Provisional Patent Application Ser. No. 61/093,661, filed Sep. 2,2008. The entire text of each of the above referenced disclosures isspecifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of oncology andchemotherapy. More particularly, it concerns the use of 2,4-disulfonylphenyl tert-butyl nitrone (2,4-ds-PBN), alone or in combination withchemo- or radiotherapy, to treat gliomas.

2. Description of Related Art

Gliomas are a diverse group of brain tumors that arise from normal“glial” cells of the brain and/or their precursor cells. The mostimportant determinant of survival for gliomas is the “grade” of theglioma. Secondary determinants of survival are age at diagnosis,performance status, and extent of surgery. Patients with low-gradegliomas have a protracted natural history with generally long survivaltimes, while those with high grade gliomas are much more difficult tosuccessfully treat and have shorter survival times. All gliomas havespecific signs and symptoms that are primarily related to the locationand size of the glioma.

The temporal lobe gliomas, for example, may cause seizures, difficultywith speech and/or loss of memory. The frontal lobe gliomas may causeseizures, behavioral changes, weakness of the arms or legs on theopposite side of the body, and/or difficulty with speech. The occipitalgliomas may cause loss of vision. The parietal gliomas may cause loss ofspatial orientation, diminished sensation on the opposite side of thebody, and/or inability to recognize once familiar objects or persons.

Astrocytomas are glioma tumors that arise from brain cells calledastrocytes or their precursors. Astrocytes are cells in the centralnervous system that support neuronal function. Astrocytomas can begraded by histologic features that signify increasing malignancy intoastrocytoma, anaplastic astrocytoma, or glioblastoma multiforme.Anaplastic astrocytoma and glioblastoma multiforme are consideredhigh-grade gliomas while the astrocytoma is considered to be a low-gradeglioma. High-grade tumors grow rapidly and can easily infiltrate andspread through the brain. Low-grade astrocytomas can also infiltrate thebrain but are usually more localized and grow slowly over a long periodof time. High-grade tumors are much more aggressive and require veryintense therapy. The majority of astrocytic tumors in children arelow-grade, whereas the majority in adults are high-grade. Astrocytomascan occur anywhere in the brain and spinal cord, however the majorityare located in the cerebral hemispheres.

Oligodendrogliomas are also gliomas. They arise from oligodendrocytesand/or their cell precursors. Normal oligodendrocytes provide myelin, afatty substance that covers nerve axons in the brain and spinal cord andallows nerves to conduct electrical impulses more efficiently.Oligodendrogliomas are classified as low grade oligodendroglioma (lessaggressive) and anaplastic oligodendroglioma (more aggressive). Morecommon than pure oligodendrogliomas are low grade and anaplastic tumorsthat are a mixture of astrocytoma and oligodendroglioma(“oligoastrocytomas”).

Anaplastic oligodendrogliomas and mixed oligoastrocytomas are moresensitive to cytotoxic chemotherapy than astrocytomas. A high rate ofresponse to the PCV (procarbazine (matulane), CCNU (lomustine),vincristine) chemotherapy has made the use of this regimen, if not thestandard of care for these tumors, at least a very common treatment. Lowgrade oligodendrogliomas are also sensitive to chemotherapy, and PCV canbe used when low grade tumors begin to grow despite priorsurgery/radiation therapy.

In 1983, it was reported that surgery plus radiation therapy and BCNUchemotherapy significantly improved the survival of patients withmalignant glioma, as compared to those treated with surgery plusradiation therapy without chemotherapy. In one study, both procarbazineand streptozotocin demonstrated effectiveness similar to that of BCNU.Other studies showed that BCNU alone is as effective as BCNU followed byprocarbazine, and that PCNU was no better than BCNU. In some studies thePCV combination was found superior to BCNU for anaplastic gliomas, whilein other studies they are considered equivalent.

Another approach involves the combined use of DFMO-PCV in treatinganaplastic gliomas, extending the study of Levin et al. (2000).Surprisingly, and in marked contrast to the results seen withglioblastoma multiforme, the combination of DFMO and PCV significantlyincreased the overall survival of patients with anaplastic gliomas. Itis possible that a feature distinguishing tumor response to DFMO is thelevel and activity of the enzyme ornithine decarboxylase (ODC). It islikely that one factor that distinguishes DFMO benefit is the tumorlevel of ODC; patients with relatively lower levels of ODC appear torespond better to DFMO and DFMO-nitrosourea combinations. Thisconclusion is based on published observations that show that (1) ODClevels are directly correlated with malignant grade of glioma(Scalabrino et al., 1982; Scalabrino an Ferioli, 1985; Ernestus et al.,1992; 1996); (2) the fact that DFMO (+/− methylglyoxalbisguanylhydrazone) activity was not seen in glioblastoma multiforme andbest seen in mid-grade anaplastic gliomas (Levin et al., 1992; 1995)with lower ODC levels; and (3) in combination with a nitrosourea,activity was not seen in glioblastoma multiforme and best seen inmid-grade anaplastic gliomas (Prados et al., 1989; Levin et al., 2000)with lower ODC levels.

The inventors have demonstrated the efficiency ofphenyl-tert-butyl-nitrone (PBN) as a potential anti-glioma drug in thepre-treatment of a rat C6 glioma implantation model (Doblas et al.,2008). MR results from untreated rats showed the diffusive invasivenessof C6 gliomas, with some associated angiogenesis. PBN administration asa pretreatment was found to clearly induce a decrease in growth rate andtumor regression as well as preventing angiogenesis. However,post-treatment of PBN had little effect for tumor regression comparedwith pre-treatment. MR findings rivaled those from histology andangiogenesis marker immunostaining evaluations.

In addition to controlled survival-based clinical trials, a large numberof other agents have been tested in response-based studies in gliomapatients. Regardless, there is a need for effective and improvedtherapies for anaplastic gliomas.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod for treating a glioma in a subject comprising administering tosaid subject with glioma a dose of 2,4-disulfonyl derivative ofphenyl-tert-butyl nitrone effective to inhibit the growth of saidglioma. The method human subject may have a recurrent or metastaticglioma, or have previously failed one or more anti-glioma therapies. Theeffective dose may be from about 5 to about 150 mg/kg body weight perday. Administering may be through dietary administration, such asthrough supplementation of a food component. The effective amount isfrom about 0.005 w/w % to about 0.1 w/w % of the diet beingadministered. The glioma may be an astrocytoma, an oligodendroglioma, ora glioblastoma multiforme. The method may also further comprise asecondary anti-glioma therapy, such as chemotherapy, including islomustine, vincristine, matulane, PCV, BCNU, CCNU and/or DFMO, radiationor surgery. Treating may comprise inhibiting the vascularization, growthor spread of a glioma, reducing tumor burden, or preventing metastasis.The subject may be an non-human animal subject or a human.

In another embodiment, there is provided a method for inhibiting gliomadevelopment comprising (a) identifying a subject at risk of developing aglioma and (b) administering to said subject a dose of 2,4-disulfonylderivative of PBN or phenyl-tert-butyl nitrone effective to inhibit thedevelopment of said glioma. The subject may have a familial history ofcancer or may have been exposed to a carcinogenic environment. Specificglioma risk factors include exposure to N-nitroso compounds orX-irradiation. The effective dose is from about 5 to about 150 mg/kgbody weight per day. Administering may be through dietaryadministration, such as through supplementation of a food component,oral administration in the form of a pill or in liquid form, or viaintravenous injection. The effective amount may be from about 0.005 w/w% to about 0.1 w/w % of the diet being administered. The glioma may bean astrocytoma, an oligodendroglioma, or a glioblastoma multiforme. Thesubject may be a non-human animal subject or a human.

In yet another embodiment, there is provided a method for inhibitingglioma recurrence comprising administering to a subject previouslyhaving a glioma a dose of 2,4-disulfonyl derivative of PBN orphenyl-tert-butyl nitrone effective to inhibit the development of saidglioma. The glioma may be an astrocytoma, an oligodendroglioma, or aglioblastoma multiforme. The effective dose may be from about 5 to about150 mg/kg body weight per day. Administering may be through dietaryadministration, such as through supplementation of a food component,oral administration in the form of a pill or in liquid form, or viaintravenous injection. The effective amount is from about 0.005 w/w % toabout 0.1 w/w % of the diet being administered. The subject may be annon-human animal subject or a human. The method may further compriseadministering a second agent that inhibits glioma occurrence. The methodmay also further comprise screening for glioma formation in saidsubject.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, adevice or a method that “comprises,” “has,” “contains,” or “includes”one or more elements possesses those one or more elements, but is notlimited to possessing only those one or more elements or steps.Likewise, an element of a device or method that “comprises,” “has,”“contains,” or “includes” one or more features possesses those one ormore features, but is not limited to possessing only those one or morefeatures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-C: MRI (T1-weighted axial slice in mid-brain region) of C6glioma at days 10 (FIG. 1A), 15 (FIG. 1B) and 18 (FIG. 1C) after cellinjection. Note tumor outlined by arrows.

FIG. 2-1( a)-2-2(d): H&E staining (1) and von Willebrand factorimmunostaining (2) of a C6 glioma at 23 days. Areas of normal tissue(×40; a), glioma (×40; b), necrosis (×40; c) and infiltration (×10; d)are presented (Doblas et al., 2008).

FIG. 3: Parent nitrone, PBN, and sulfonyl derivatives, 2-S-PBN and2,4-ds-PBN.

FIG. 4: MR images of 2,4-ds-PBN- and non-treated rats with C6 gliomas(post-tumor>15 days). Tumors are hyperintense regions (outlined). Imagesat day 16 (d16), d20 and d25.

FIG. 5: (Left panel) Tumor volumes measured from MR images of rats withC6 gliomas treated with 2,4-ds-PBN (18 mg/kg/day in drinking water)compared to untreated rats. (Right panel) Percent survival of rats withC6 gliomas treated with 2,4-ds-PBN compared to untreated rats.

FIG. 6: Western blots of rat C6 cells with increasing concentrations of2,4-ds-PBN. Increasing levels of apoptotic factors pro-caspase 3 andpro-caspase 9 are shown, relative to actin.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 1. The Present Invention

The prognosis of patients who are diagnosed with glioblastoma multiformeis very poor, due to the difficulty of an early and accurate diagnosisand the lack of currently efficient therapeutic compounds. Using aglioma model previously applied to the testing of PBNs and non-sulfonylderivatives thereof, the inventors examined the effect of a nitronecompound, 2,4-ds-PBN that is a structural analog of PBN. Magneticresonance results from 2,4-ds-PBN post-treated rats indicated apparentdecrease in tumor volume and the delay in tumor growth rate. 2,4-ds-PBNpost-treatment was also significantly effective in increasing survivalrate.

This result was somewhat unexpected and surprising as sulfonatedderivatives of PBN are known not to be able to readily cross theblood-brain-barrier (BBB). PBN, the parent nitrone, has been previouslyfound to easily penetrate the BBB (Wang & Shuaib, 2007). 2,4-disulfonylPBN (2,4-ds-PBN) is structurally related to the parent compound PBN, butcontains two sulfonyl groups that make it much more water-soluble. As aresult of the increased water solubility 2,4-disulfonyl PBN cannoteasily pass through the BBB, as compared to PBN (Wang & Shuaib, 2007).Ideally, drugs that are to be used as anti-glioma therapy need to passthe endothelial junctions of the BBB to reach the majority of tumorcells (Cao et al., 2005). It is possible that malignant gliomas haveacquired the ability to actively degrade tight junctions by secretingsoluble factors, eventually leading to BBB disruption within invadedbrain tissue (Schneider et al., 2004). Regardless, it has never beenthought that 2,4-disulfonyl PBN would reach sufficient amounts in gliomatissue to have any therapeutic effect. For instance RenovisPharmaceuticals, administered 2,4-disulfonyl PBN as an anti-stroketherapeutic agent via i.v. administration to concentrate this compoundinto brain tissue (Shuaib et al., 2007).

These and other aspects of the invention are described in detail below.

2. Anaplastic Glioma Strata

A. Clinical Features

The anaplastic gliomas are intermediate grade infiltrativegliomas—classified between low (localized, slow growing) andglioblastoma multiforme (rapidly growing and highly invasive).Anaplastic astrocytomas (AA) are tumors that arise from brain cellscalled astrocytes and/or their precursors. Astrocytes are support cellsof the central nervous system. The majority of astrocytic tumors inchildren are low-grade, whereas the majority in adults are high-grade.These tumors can occur anywhere in the brain and spinal cord.

Oligodendrogliomas are gliomas derived from oligodendrocytes and/ortheir precursors. Oligodendrocytes that have a role in the structure andfunction of myelinated neurons in the brain. Anaplasticoligodendroglioma (AO) are more aggressive than oligodendrogliomas, butare also more sensitive to chemotherapy than are anaplasticastrocytomas. A high rate of response to the use of PCV (procarbazine,CCNU, vincristine) chemotherapy has led to the common use of PCVchemotherapy prior to radiation therapy, following irradiation, and/orat tumor recurrence and progression. Another glioma appears ashistologic mixture of both oligodendroglioma and astrocytoma tumor formsand is called oligoastrocytoma. While oligoastrocytoma can be low-grade,the majority of the mixed oligoastrocytomas are anaplasticoligoastrocytomas (AOA).

The last glioma subgroup are ependymomas. One subtype of malignantependymomas is the anaplastic ependymoma (AE); these tumors arise fromependymal cells and/or their precursors that line the cerebrospinalfluid passageways, called ventricles. These tumors are classified aseither supratentorial (in the top part of the head) or infratentorial(in the back of the head).

Clinical features and symptoms produced by gliomas depend on thelocation of the tumor and the age of the patient. The most commonlocation for gliomas is in the cerebral hemispheres in adults and thecerebellum, brainstem, hypothalamus, and thalamus in children. Spinalcord gliomas are much less common then gliomas of the brain. Patientswith these tumors have symptoms that vary depending on location in thebrain or spinal cord. They can produce symptoms of headache, seizures,nausea and vomiting, limb weakness, unilateral sensory changes,personality change, and unsteadiness in walking.

B. Classifications

Anaplastic Astrocytoma.

The histologic features of anaplastic astrocytomas are similar to thoseof low-grade astrocytomas but these features are more abundant andexaggerated. These tumors are WHO grade III (Kleihues et al., 1993;Kleihues and Cavenee, 2000). Cellularity is more increased, as arenuclear and cellular pleomorphism. These features may be extreme, withback-to-back cells and bizarre, hyperchromatic nuclei. Cytoplasm may bescanty, with nuclear lobation and enlargement indicating anaplasia.Mitotic activity is easily recognized in most anaplastic astrocytomasbut inexplicably may be absent in areas with gemistocytes.

The range of anaplasia in this grade is broad, with some examplesshowing low cellularity and pleomorphism with a few mitotic figures andothers being highly cellular and pleomorphic with frequent mitoses,lacking only the necrosis required for a histologic diagnosis ofglioblastoma. For this reason, it is useful to have a more objectiveindicator of behavior, and some markers of cell proliferation have beenused in an attempt to predict prognosis more accurately. The most usedmarkers in this area have been antibodies to bromodeoxyuridine (BrdU)and Ki-67 (Davis et al., 1995). The cellular incorporation of BrdU is aspecific marker of the DNA synthesis phase of the cell cycle, whereasthe Ki-67 antibody labels an antigen that is present in all phases ofthe cell cycle except G₀. Both antibodies can be identified byimmunohistochemical staining in paraffin-embedded tissue sections. As ageneralization, higher labeling rates for anaplastic astrocytomas isassociated with poor prognosis (Hoshino et al., 1993; Davis et al.,1995; Lamborn et al., 1999).

Glioblastoma Multiforme.

Glioblastoma, also known as glioblastoma multiforme, is the glioma withthe highest grade of malignancy, WHO grade IV (Kleihues and Cavenee,2000). It represents 15% to 23% of intracranial tumors and about 50%-60%of astrocytomas. Most examples are generally considered to arise fromastrocytes because glial fibrillary acidic protein can be identified inthe cell cytoplasm. Some examples, however, apparently arise from otherglial lineages, such as oligodendrocytes. Glioblastoma is the mostfrequently occurring astrocytoma. Autopsy and serial biopsy studies haveshown that some astrocytomas progress through the grades of malignancywith transformation from low-grade to anaplastic astrocytoma toglioblastoma (Muller et al., 1977). But, because some examples ofglioblastoma appear to arise rapidly in otherwise normal patients andare recognized when they are small, it is thought that this variety ofglioblastoma can also arise directly from malignant transformation ofastrocyte precursor cells without passing through the lower grades ofmalignancy (Kleihues and Ohgaki, 1997; 1999).

Tumor necrosis is the characteristic gross feature that distinguishesglioblastoma from anaplastic astrocytoma (Nelson et al., 1983; Burger etal., 1985; 1991). Another microscopic feature that is distinctive anddiagnostic is the presence of proliferative vascular changes within thetumor. These changes may occur in the endothelial cells (vascularendothelial hyperplasia or proliferation) or in the cells of the vesselwall itself (vascular mural cell proliferation). Both types of changeare sometimes considered together as microvascular proliferation.Glioblastomas cellularity is usually extremely high. The individualcells may be small, with a high nuclear:cytoplasmic ratio, or very largeand bizarre, with abundant eosinophilic cytoplasm. These same smallcells may appear to condense in rows around areas of tumor necrosis,forming the characteristic pseudopalisades. Glioblastoma tumors have apropensity to infiltrate the brain extensively, spreading even todistant locations and giving the appearance of a multifocal glioma. Someexamples are truly multifocal (i.e., arising in multiple simultaneousprimary sites) while many of these multifocal tumors show a histologicconnection when the whole brain is examined at autopsy.

Oligodendrogliomas.

Like astrocytomas, oligodendrogliomas mimic the histology of theirpresumed cell of origin. They also arise primarily in the white matterbut tend to infiltrate the cerebral cortex more than do astrocytomas ofa similar grade of malignancy. Like astrocytomas, grading schemes ofhistologic malignancy have been used for oligodendrogliomas, but thesecorrelate less well with prognosis than those used for astrocytomas(Burger et al., 1987; Bigner et al., 1998; Daumas-Duport et al., 1997).Many of the histologic features used to grade oligodendrogliomas aresimilar to those used for astrocytomas: cellularity, pleomorphism,mitotic activity, vascular changes, and necrosis. Lower-gradeoligodendrogliomas may have microcysts. Oligodendrogliomas of allhistologic grades tend to infiltrate the cortex readily and to formclusters of neoplastic cells in the subpial region, around neurons, andaround blood vessels. In general, the cells of oligodendrogliomas haveround, regular nuclei and distinct cytoplasmic borders with clearing ofthe cytoplasm. Another fairly distinctive and diagnostically helpfulfeature is the vascular pattern of oligodendrogliomas, referred to as“chicken-wire” vessels that can divide the tumor into discrete lobules.With increasing anaplasia, oligodendrogliomas can become highly cellularand pleomorphic, approaching an appearance of glioblastoma multiformewith the presence of necrosis. Although it is correct to classify theseas anaplastic oligodendrogliomas, some would use the term glioblastomaonce necrosis is identified in any high-grade glial neoplasm. Onejustification for separating anaplastic oliogdendrogliomas fromastrocytic glioblastomas is the slightly better prognosis of the former,even in this highest grade of malignancy. Some authors have reportedthat a MIB-1 labeling index of >3%-5% predicts a worse prognosis inoligodendrogliomas (Heegard et al., 1995; Kros et al., 1996; Dehghani etal., 1998).

Oligoastrocytomas.

Many, if not most, oligodendrogliomas occur with a regional or intimatecellular mixture of astrocytoma. For the diagnosis of mixed glioma, theproportion of each should be substantial, but authors have differingopinions with respect to exact numbers; usually a mixture with a rangefrom 10% to 25% of the minor element is used to diagnose a mixed glioma.Oligoastrocytomas and anaplastic oligoastrocytomas correspond to WHOgrade II or grade III, respectively (Kleihues and Cavenee, 2000).Histologic features of anaplasia may be present in either component andwill affect the prognosis adversely. Such features include markedcellular pleomorphism, high cellularity, and a high mitotic rate.Microvascular proliferation and necrosis may also be seen. Prognosis andresponse to therapy have not been shown to depend on the proportion ofthe oligodendroglial versus the astrocytic component (Shaw et al.,1994), although paradoxically, the BrdU LI of the oligodendroglialcomponent is more predictive for survival than the astrocytic component(Wacker et al., 1994) and far advanced tumor progressions are dominatedby the astrocytic component.

3. Phenyl N-Tert-Butyl Nitrone (PBN)

A. PBN's

The compound phenyl N-tert-butyl nitrone (PBN) was first synthesized inthe 1950's, but in 1968 it was discovered to be very useful to trap andstabilize free radicals in chemical reactions and hence it was termed aspin-trap (Janzen, 1971). Although PBN is the prototype spin-trap,several other nitrones have been synthesized and found useful to trapand characterize free radicals in chemical reactions. These spin trapswere used in chemical reactions first, but in the mid-1970's they beganto be used to trap free radicals in biochemical and biological systems(Floyd et al., 1977; Poyer et al., 1978). Pharmacokinetic studies haveshown that PBN is readily and rapidly distributed almost equally to alltissues, has a half-life in rats of about 132 minutes and is eliminatedmostly in the urine. Relatively few metabolism studies have been done,but it is known that some ring hydroxylation (primarily in the paraposition) of the compound occurs in the liver.

Novelli first showed that PBN could be used to protect experimentalanimals from septic shock (Novelli et al., 1986), and indeed this waslater confirmed by other groups (Pogrebniak et al., 1992). The use ofPBN and derivations as pharmacological agents began after discoveries in1988 that showed that PBN had neuroprotective activity in experimentalbrain stroke models (Floyd, 1990; Floyd et al., 1996; Carney et al.,1991). These results were repeated and extended, (see Clough-Helfman etal., 1991; Cao et al., 1994; Folbergrova et al., 1995; Pahlmark et al.,1996). Others inventors have summarized the extensive neuroprotectivepharmacological research effort on PBN and derivatives (Floyd, 1997;Hensley et al., 1996). In addition to neurodegenerative diseases, PBNhas been shown to protect in other pathological conditions whereROS-mediated processes are involved, including diabetes and many otherconditions. The mechanistic basis of why PBN and some of its derivativesare so neuroprotective in experimental stroke and several otherneurodegenerative models has not been completely elucidated yet.However, it is clear that its action cannot simply be explained by itsability to trap free radicals.

The general formula for PBNs is:

wherein:

X is phenyl or

R is H,

and n is a whole integer from 1 to 5; or

Y is a tert-butyl group that can be hydroxylated or acetylated on one ormore positions;phenyl; or

wherein W is

or Z; and Z is a C₁ to C₅ straight or branched alkyl group.

B. PBN's in Cancer

U.S. Pat. No. 5,569,902 (incorporated herein by reference) describes theuse of nitrone free radical trapping agents for the treatment of cancer.Specifically, PBN and related compounds are described as being useful inthe preparation of an anti-carcinogenic diet and the preparation of suchsupplemented diets. Those subjects most likely to beneficially receivethe nitrones would include: (1) those having had pretumor testsindicating a high probability of the presence of tumors, (2) thoseexposed to very potent carcinogenic environments and their probabilityof tumor progression is high, and (3) to those whose geneticpredisposition makes their likelihood of tumor development high.

U.S. Patent Publication 2007/0032453 (incorporated herein by reference)describes the effect of the anti-inflammatory phenyl N-tert-butylnitrones (PBNs) on gliomas using MRI techniques. PBN itself was able tocontrol tumor development when provided to a subject either before, atthe time of or after tumor implantation. Thus, it was proposed to usePBN, and related nitrone free radical trapping agents, as therapeuticagents for gliomas.

C. 2,4-Disulfonyl Phenyl N-Tert-Butyl Nitrone (2,4-Ds-PBN)

U.S. Pat. No. 5,488,145 (incorporated herein by reference) describes2,4-disulfonyl phenyl-tert-butyl nitrone and its pharmaceuticallyacceptable salts. These materials were described as usefulpharmaceutical agents for oral or intravenous administration to patientssuffering from acute central nervous system oxidation as occurs in astroke or from gradual central nervous system oxidation which canexhibit itself as progressive central nervous system function loss.

2,4-disulfonyl PBN's two sulfonate groups was expected to exhibitimproved water solubility, but was also expected to exhibit poortransport across the blood/brain barrier because of its lipophobiccharacter. However, when the present compound was made and tested invivo, it showed an unexpected increase in efficacy as compared to PBN.This increase in efficacy occurred along with an increase in potency ascompared to PBN. In direct contrast to this marked increase in potencyand efficacy there was a marked and highly significant decrease intoxicity as compared to PBN.

These results were unexpected because in the general literature onstructure/activity relationships within specific defined families ofcompounds therapeutic potency typically covaries with toxicity. Thus,most related compounds maintain their ratio of therapeutic potency totoxicity. In contrast, the compound of this invention deviates from thisexpected relationship when its potency increased and its toxicitydecreased relative to closely related analogs.

Accordingly, in one aspect, the invention provides the PBN-disulfonylcompound and its pharmaceutically acceptable salts. In a second aspect,the invention provides intravenously- and orally-administrablepharmaceutical compositions having this compound or its salt as activeingredient.

2,4-ds PBN may exists at higher pHs in an ionized salt form:

where X is a pharmaceutically acceptable cation. Most commonly, thiscation is a monovalent material such as sodium, potassium or ammonium,but it can also be a multivalent alone or cation in combination with apharmaceutically acceptable monovalent anion, for example calcium with achloride, bromide, iodide, hydroxyl, nitrate, sulfonate, acetate,tartrate, oxalate, succinate, palmoate or the like anion; magnesium withsuch anions; zinc with such anions or the like. When these combinationsof a polyvalent cation and a monovalent anion are illustrated instructural formulae, herein, the monovalent anion is identified as “Y.”

Among these materials, the free acid and the simple sodium, potassium orammonium salts are most preferred with the calcium and magnesium saltsalso being preferred but somewhat less so.

2,4-ds PBN can be prepared by a two-step reaction sequence. In the firststep, commercially available tertiary butyl nitrate(2-methyl-2-nitropropane) is converted to the correspondingn-hydroxylamine using a suitable catalyst such as an activatedzinc/acetic acid catalyst or an aluminum/mercury amalgam catalyst. Thisreaction can be carried out in 0.5 to 12 hours and especially about 2 to6 hours or so at a temperature of about 15-100° C. in a liquid reactionmedium such as alcohol/water mixture in the case of the zinc catalyst oran ether/water mixture in the case of the aluminum amalgam catalyst.

In the second step, the freshly formed hydroxylamine is reacted with4-formyl-1,3-benzenedisulfonic acid, typically with a slight excess ofthe amine being used. This reaction can be carried out at similartemperature conditions. This reaction is generally complete in 10 to 24hours.

The product so formed is the free acid and is characterized by amolecular weight of 89 g/mole. It is a white powdery material whichdecomposes upon heating. It is characterized by a solubility in water ofgreater than 1 gram/ml and a ¹H NMR spectrum in D₂O of 8.048 ppm (dd,8.4, 1.7 Hz); 8.836 ppm (d, 8.4 Hz); 8.839 ppm (d, 1.7 Hz); 8.774 ppm(s).

The various salts can be easily formed by admixing the free acid inaqueous medium with two equivalents of the appropriate base, forexample, KOH for the potassium salt, and the like.

One synthesis is based on the work by R. H. Hinton and E. G. Janzen (J.Org. Chem. 57:2646-2651, 1992). It involves the condensation of analdehyde with a hydroxylamine. The hydroxylamine is unstable and isprepared fresh on the day of use using an activated zinc catalyst. Thesynthesis is as follows.

TABLE 1 Prerequisite Chemicals 1. 95% Ethanol 2. 2-Methyl-2-nitropropane3. Zinc dust 4. Glacial acetic acid 5. Diethyl ether 6. Saturated sodiumchloride 7. Magnesium Sulfate, Anhydrous solid 8.4-Formyl-1,3-benzenesulfonic acid (MW 310.21 g/mole), disodium salt,hydrate 9. Methanol 10. Dichloromethane

TABLE 2 Preparation of N-t-Butylhydroxylamine 1. A 500 mL three neckround bottom flask is equipped with a magnetic stir bar, thermometeradapter, thermometer, and addition funnel. 2. 95% ethanol (350 mL) wasadded to the flask and cooled to 10° C. in an ice bath. 3.2-Methyl-2-nitropropane (6.18 g, 0.060 mole), and zinc dust (5.89 g,0.090 mole) were added in single portions. 4. Glacial acetic acid (10.8g, 0.180 mole) was placed in the addition funnel and added dropwise atsuch a rate with vigorous stirring to maintain the temperature below 15°C. 5. The ice bath was removed and mixture was stirred for 3 hrs at roomtemperature. 6. The solvent was stripped from the mixture, leavingt-butylhydroxyl- amine, zinc acetate, and water. 7. Dichloromethane (50mL) was added and the mixture filtered through a Buchner funnel. 8. Thezinc acetate cake left on the filter paper was washed with 2X 25 mLdichloromethane. 9. Water was separated from the filtrate in aseparatory funnel and the organic layer dried over magnesium sulfate.10. The magnesium sulfate was removed by filtering through fluted filterpaper, then dichloromethane stripped off by rotary evaporation. 11. Theproduct (100% yield = 5.34 g), viscous liquid, was dissolved in methanol(50 mL) for use below.

TABLE 3 Preparation of 2,4-disulfonylphenyl-N-t-butylnitrone 1. A 3-neck250 ml round bottom flask was set up with a stir bar, a gas dispersiontube, an addition funnel, and a Friedrichs condenser cooled withrecirculating ice water. 2. To the flask were added 200 mL of methanol,4-formyl-1,3-benzenedisulfonic acid (9.31 g, 30 mmoles) andN-t-butylhydroxylamine (25 mL of the methanol solution from part A, 30mmoles theoretical). 3. The reaction was heated to reflux with a heatingmantle while bubbling the reaction with nitrogen with stirring. 4. Themixture was refluxed for 2 hours. 5. The remainder of hydroxylamine fromabove was added. 6. Refluxing was continued with nitrogen bubbling forat least 18 hours, but not more than 24 hours. 7. The hot reactionmixture was filtered on a Buchner funnel, and the solid washed with hotmethanol. 8. The methanol was stripped off by rotary evaporation to ayellow, viscous oil. 9. Hot 1:1 ethanol:acetone (200 mL) was added andthe mixture heated to dissolve the oil. 10. The solution was cooled tocrystallize the product. 11. The product was collected on a Buchnerfunnel and dried under vacuum overnight. 12. The reaction typicallygives 75% yield of I, a white powder. Other methods of synthesis aredisclosed in the prior art as well.

4. Combination Treatments

In one embodiment, 2,4-ds-PBN may be used in conjunction with anotherglioma therapy, such as radiation, PCV, DFMO, CCNU or BCNU. Thesecompositions would be provided in a combined amount effective to kill orinhibit proliferation of the cell. This process may involve contactingthe cells with the agents at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes 2,4-ds-PBN and the other includes the second agent.

Alternatively, the 2,4-ds-PBN therapy may precede or follow the otheragent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and 2,4-ds-PBN are applied separatelyto the cell, tissue or organism, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agents would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with both modalities withinabout 12-24 h of each other and, more preferably, within about 6-12 h ofeach other. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several d (2, 3, 4,5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Multiple administrations of each agent are contemplated. For example,where the 2,4-ds-PBN therapy is “A” and the secondary agent or therapyis “B,” the following are contemplated:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Patients will be evaluated for neurological changes considered to beindependent of tumor and graded using NCI Common Toxicity Criteria(neurotoxicity). Aside from baseline audiometric testing, repeataudiometric testing for ototoxicity is performed at the physician'sdiscretion for patients who had evidence of hearing loss or progressionof hearing loss by neurological examination. In addition, blood countsare performed biweekly, and serum creatinine, alkaline phosphatase,bilirubin and alanine amino-transferase tests are performed before eachcycle. Doses may be modified during the course of treatment, primarilybased on neutrophil and platelet counts (vincristine, lomustine andmatulane) or ototoxicity (DFMO). Occasionally, DFMO dose reductions arerequired for diarrhea.

A. PCV

PCV is a drug combination therapy employing three different agents—ahydrazine derivative, matulane, a nitrosourea, lomustine, and a tubulininteractive agent, vincristine. It has been used in a number of clinicaltrials, most notably by the inventor in assessing its effect onhigh-grade glioma and medulloblastoma tumors. The major side-effectobserved with PCV was dose-limiting myelotoxicity. Each of thecomponents of PCV is described below.

It should be noted that the present invention could include the use ofBCNU rather than of CCNU (lomustine) since both are nitrosoureas. Italso is contemplated that one could use CCNU and procarbazine or BCNUand procarbazine, without vincristine, since vincristine is usuallyconsidered to be the least active of the drugs in the PCV combination.

Both hydrazines and nitrosoureas are alkylating agents. As a group,alkylating agents form covalent chemical adducts with cellular DNA, RNAand protein molecules and with smaller amino acids, glutathione andsimilar chemicals. Generally, these alkylating agents react with anucleophilic atom in a cellular constituent, such as an amino, carboxyl,phosphate, sulfhydryl group in nucleic acids, proteins, amino acids, orglutathione. The mechanism and the role of these alkylating agents incancer therapy is not well understood. In addition to hydrazine andnitrosoureas, alkylating agents include: triazenes such as dacarabzineand temozolomide, nitrogen mustards such as chlorambucil,cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard;aziridine such as thiotepa; methanesulphonate esters such as busulfan;platinum complexes such as cisplatin, carboplatin; bioreductivealkylators, such as mitomycin and altretemine. Any of these compoundsmay be used together or individually, in combination with the compoundsof the present invention.

i. Hydrazine and Triazene Derivatives

Hydrazine and triazene derivatives are similar to nitrosoureas in thatthey decompose spontaneously or are metabolized to produce alkylcarbonium ions, which alkylate DNA. This class of compounds includesmatulane, dacarbazine and temozolomide.

The active ingredient in matulane is Procarbazine Hydrochloride(N-isopropl-alpha-(2-methylhydrazino)-p-toluamide monohydrochloride). Itis available from Roche Laboratories, Inc. It was approved in 1969 fortreatment of Hodgkins' Disease. The typical form is an oral capsule thatcontains 50 mg procarbazine as the hydrochloride. Dosages vary dependingupon whether procarbazine is being used as a combination drug with otheranticancer drugs or as a single therapeutic agent. A suggested guidelineper the PDR for single agent use is 100 mg two times daily for 14 days.

The exact mode of actions of matulane is not clear. There is someevidence that the drug acts by inhibition of protein, RNA and DNAsynthesis. It is primarily metabolized in the liver and kidneys andappears to be auto-oxidized to the azo derivative with the release ofhydrogen peroxide. The azo derivative isomerizes to the hydrazone and,following hydrolysis, splits into a benzylaldehyde derivative andmethylhydrazine. The methylhydrazine is further degraded to CO₂ and CH₄,and possibly hydrazine, whereas the aldehyde is oxidized to acid whichis excreted in the urine.

Matulane exhibits monamine oxidase inhibitory activity (MAOI), so a dietthat restricts foods which contain high tyramine content should befollowed. Drugs to be avoided during therapy include antihistamines,sympathomimetics, barbiturates, narcotics, hypotensive agents orphenothiazines, and ethyl alcohol. Some foods are also to be avoidedduring procarbazine such as naturally aged cheeses, chocolates, nuts,and bananas as they could theoretically lead to a hypertensivecomplication in some patients. Also, unacceptable toxicity may occur ifmatulane is used in patients with impairment of renal and/or hepaticfunction. Treatment may be curtailed in the event of central nervoussystem signs or symptoms such as paresthesias, neuropathies orconfusion; neutropenia (absolute neutrophil count under 1500/μl),thrombocytopenia (platelets under 100,000/μl), hypersensitivityreaction, ulceraction or persistent spot of soreness around the oralcavity, diarrhea or loose stools, hemorrhage or bleeding tendencies.

Adverse but expected reactions include leukopenia, neutropenia, anemia,and thrombocytopenia. Commonly reported acute side effects are nauseaand vomiting during or shortly after dose administration.

ii. Nitrosoureas

Nitrosoureas represent a group of therapeutic alklyating agents. Thisclass of compounds includes lomustine, carmustine, semustine,steptozocin, and nimustine.

(a) Lomustine

Lomustine is a synthetic alkylating agent, also known as CCNU, with thechemical name of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. It wasapproved in 1977 for treatment of brain tumors and Hodgkin's Disease. Itis available from Bristol Myers Squibb as oral capsule, available in 10mg, 40 mg and 100 mg forms. Dosages may vary depending upon whetherlomustine is being used as a single agent or in a combination inaddition to other chemotherapeutic agents. As a single agent inpreviously untreated patients, the recommended dosages per the PDR is130 mg as a single oral dose every 6 weeks. Lomustine crosses the bloodbrain barrier.

It is believed that CCNU alkylates DNA and RNA. It is cross-resistantwith other nitrosoureas and some but not all alkylating agents. It mayalso inhibit several key enzymatic processes by carbamoylation of aminoacids in proteins.

The most common and severe toxic side effects are bone marrowsuppression leading to thrombocytopenia and leukopenia, which maycontribute to bleeding and infections. Bone marrow toxicity iscumulative and thus dosage adjustments must be considered on the basisof the nadir blood counts from prior doses.

(b) Carmustine

Carmustine, also known as BCNU, with the chemical name ofN,N-Bis(2-chloroethyl)-N-nitrosurea, is a nitrosurea alkylating agentapproved by the FDA in 1977. Carmustine has ben used for many years fortreatment of primary brain tumors and is used for the treatment ofgliomas. Carmustine is available from Bristol Meyers Squibb in packagescontaining vials of 10 mg carmustine and 3 ml sterile diluent fordelivered by i.v. injection. As a single agent carmustine isadministered at about 150-200 mg/m² every 6 weeks. In combinationregimens, carmustine may be given in does similar to those of lomustine.An alternative mode of delivery is by wafers implanted directly into thetumor site (Gliadel® Wafer).

Potential side effects include bone marrow suppression, anemia,diarrhea, low white blood cell and platelet counts, pulmonary toxicityand swallowing difficulties.

iii. Tubulin Interactive Agents

Tubulin interactive agents interfere with cell division by binding tospecific sites on Tubulin, a protein that polymerizes to form cellularmicrotubules. Microtubules are critical cell structure units. When theinteractive agents bind on the protein, the cell cannot properly formmicrotubules. Tubulin interactive agents include vincristine andvinblastine, both alkaloids and the taxanes, such as paclitaxel anddocetaxel.

Vincristine, is available as Oncovin™ from Eli Lilly & Company and asVincristine Sulfate from Faulding. Also called vincaleukoblastine, a22-oxo-, sulfate (1:1) (salt), the salt of an alkaloid obtained from acommon flowering herb, the periwinkle plant. It is delivered byintravenous injection. It was approved in 1963 on label for Ewing'sSarcoma, rhabdomyosarcoma, Wilm's Tumor, neuroblastoma, Hodgkin'sDisease and leukemia.

The mechanism of action remains under investigation; however, there isan indication that inhibition of microtobule formation in the mitoticspindle, resulting in an arrest of dividing cells at the metaphasestate, is involved. The liver is the major excretory organ. Most of anintravenous dose of Vincristine is excreted into the bile after rapidtissue binding. Vincristine does not appear to cross the blood brainbarrier.

Vincristine has been reported to reduce blood levels of antiseizuremedications and to increase seizure activity. The most common adversereaction is hair loss. Leukopenia, neuritic pain and constipation occur,but usually for less than 7 days.

B. DFMO

Numerous highly proliferative types of cancer are associated withincreased levels of the polyamines putrescine, spermidine, and sperminein tumor tissue and blood and urine of mammals with cancer. Studies haveshown that this can be related to increased polyamine synthesis by therate-limiting enzyme, ornithine decarboxylase (ODC). The pathway forpolyamine synthesis begins with L-ornithine. This natural amino acid,although not normally incorporated into proteins, is part of the ureacycle which metabolizes arginine to ornithine and urea. Ornithine isconverted by ornithine decarboxylase (ODC) to putrescine and CO₂ and isconsidered to be the rate-limiting step in the production of polyamines.With the addition of propylamine donated from S-adenosylmethionine,putrescine is converted to spermidine. Spermidine is then converted tospermine by spermine synthetase, again in association with thedecarboxylation of S-adenosylmethionine. Putrescine, spermidine andspermine represent the three major polyamines in mammalian tissues.Polyamines are found in animal tissues and microorganisms and are knownto play an important role in cell growth and proliferation. Although theexact mechanism of the role of the polyamines in cell growth andproliferation is not known, it appears that the polyamines mayfacilitate macromolecular processes such as DNA, RNA, or proteinsynthesis. Polyamine levels are known to be high in the testes, ventralprostate, and thymus, in psoriatic skin lesions, and in other cellsundergoing rapid growth processes.

It also is well known that the rapid proliferation of tumor tissue ismarked by an abnormal elevation of polyamine levels. Hence, thepolyamines also may play an important role in the maintenance of tumorgrowth. Thus, ODC inhibitors, such as DFMO, may exert their therapeuticeffect by blocking the formation of the polyamines and thereby slowing,interrupting, or arresting the proliferation and metastases of the tumortissue.

DFMO (α-difluoromethylornithine, eflornithine, Ornidyl®) is a structuralanalog of the amino acid L-ornithine and has a chemical formulaC₆H₁₂N₂O₂F₂. DFMO can be employed in the methods of the invention as aracemic (50/50) mixture of D- and L-enantiomers, or as a mixture of D-and L-isomers where the D-isomer is enriched relative to the L-isomer,for example, 70%, 80%, 90% or more by weight of the D-isomer relative tothe L-isomer. The DFMO employed may also be substantially free of theL-enantiomer.

The dose limiting toxic effect of DFMO is thrombocytopenia (abnormallyfew platelets in the blood), which occurs in about 50% of patients,leukopenia (abnormally few leukocytes), or anemia. This toxic effect isrelatively harmless and reversible and cease upon withdrawal of thedrug.

The effect of an ODC inhibitor for the control of the growth rate ofrapidly proliferating tumor tissue has been assessed in standard animaltumor models. For example, the anti-tumor effect of DFMO has beendemonstrated in the following animal tumor models: L1210 leukemia inmice, EMT6 tumor in Balb/C mice, 7,12-dimethylbenzanthracene-induced(DMBA-induced) mammary tumor in rats, and DFMO Morris 7288C or 5123hepatoma in Buffalo rats. In addition, the anti-tumor effect of DFMO incombination with various cytotoxic agents has been demonstrated asfollows: (a) in combination with vindesine or adriamycin in L1210leukemia in mice, in Morris 7288C hepatoma in Buffalo rats, and in EMT6tumor in mice, (b) in combination with cytosine arabinoside in L1210leukemia in mice, (c) in combination with methotrexate in L1210 leukemiain mice, (d) in combination with cyclophosphamide in EMT6 tumor in miceand in DMBA-induced tumor in mice, (e) in combination with BCNU in mouseglioma 26 brain tumor, and (f) in combination with MGBG in L1210leukemia in mice, in Morris 7288C hepatoma in Buffalo rats, in P388lymphocytic leukemia in mice, and in S-180 sarcoma in mice.

Although DFMO can effectively block tumor putrescine biosynthesis, theresultant antitumor effect is cytostasis, not cytotoxicity. For example,DFMO reduces the growth rate of an MCA sarcoma, but does not producetumor regression. This finding is consistent with reports of otherinvestigators who showed that DFMO is a cytostatic agent. However,studies indicate that a significant role may exist for DFMO agents,permitting the future development of combination chemotherapeuticregimens which incorporate DFMO.

The initial promise of DFMO as a therapeutic ODC inhibitor for use inthe treatment of various neoplasias has dimmed somewhat because,although DFMO does, in fact, irreversibly inhibit ODC activity, cellstreated in vivo with DFMO significantly increase their uptake ofexogenous putrescine as described in U.S. Pat. No. 4,925,835. Theintercellular transport mechanisms of the cell do an “end run” aroundthe DFMO-impaired ODC activity by importing putrescine from theextracellular milieu. Therefore, DFMO's effect in vivo is far poorerthan in vitro. So, while DFMO treatment effectively inhibitsintracellular putrescine neogenesis, it also results in increased uptakeof extracellular putrescine, thereby offsetting its ODC inhibitoryeffect.

This problem is compounded by the fact that putrescine is present inmany common foods, such as grapefruit juice, which containsapproximately 400 ppm putrescine. This makes it virtually impossible toprovide a patient a nutritionally sufficient diet which is free ofputrescine. Therefore, DFMO-treated cells are capable of importingsufficient amounts of extracellular putrescine to support cell division.

Strategies to make DFMO more acceptable to human patients are describedin U.S. Pat. No. 4,859,452 (incorporated by reference). Formulations ofDFMO are described which include essential amino acids in combinationwith either arginine or ornithine to help reduce DFMO-inducedtoxicities.

C. Radiation

Factors that cause DNA damage and have been used extensively for cancertherapy and include what are commonly known as γ-rays, X-rays, and/orthe directed delivery of radioisotopes to tumor cells. Other forms ofDNA damaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells. The terms“contacted” and “exposed,” when applied to a cell, are used herein todescribe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

D. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery as a cancer treatment may be usedin conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor. In addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

5. Pharmaceutical Formulations

The present invention discloses numerous compositions, which in certainaspects of the invention, are administered to animals. For example,2,4-ds-PBN, as well as various secondary chemotherapeutic agents, willbe formulated for administration. Where clinical applications arecontemplated, it will be necessary to prepare pharmaceuticalcompositions of these compounds and compositions in a form appropriatefor the intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender agents suitable for introduction into a patient. Aqueouscompositions of the present invention comprise an effective amount ofthe agent, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients, such as other anti-cancer agents, can also be incorporatedinto the compositions.

Solutions of the active ingredients as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed withsurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent growth of microorganisms.Intravenous vehicles include fluid and nutrient replenishers.Preservatives include antimicrobial agents, anti-oxidants, chelatingagents and inert gases. The pH and exact concentration of the variouscomponents in the pharmaceutical are adjusted according to well-knownparameters.

An effective amount of the agents is determined based on the intendedgoal. The term “unit dose” refers to a physically discrete unit suitablefor use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired responsein association with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the subject to betreated, the state of the subject, and the protection desired. Preciseamounts of the therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual.

A. Enteral Administration

The active compounds of the present invention can advantageously beformulated for enteral administration, e.g., formulated for oraladministration. The pharmaceutical forms may include sesame oil, peanutoil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of ingestible compositions, including tables,pills and capsules. Also, it is contemplated that the agents of thepresent invention can be provided in the form of a food additive andincorporated into a daily dietary program. All of these forms aregenerally selected to be sterile and stable under the conditions ofmanufacture and storage.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, theparticular methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

B. Other Routes of Administration

In addition to the compounds formulated for enteral administration,parenteral formulations such as intravenous or intramuscular injectionare envisioned. Administration may also be nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be by intradermal,subcutaneous, or intraperitoneal injection. Also contemplated iscontinuous perfusion via catheter. Such compositions would normally beadministered as pharmaceutically acceptable compositions, describedsupra.

6. Examples

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutespecifically contemplated modes for its practice. However, those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

Example 1

The inventors used an intracerebral C6 rat glioma cell implantationmethod, widely accepted by the glioma research field, as a good modelfor gliomas. To induce glioma in rats, C6 rat glioma cells (10⁶ cells in10 μL were injected into the cerebral cortex of male Fischer 344 rats.Fifteen days later, 2,4-ds-PBN was orally administered through drinkingwater (0.015%, 17.5 mg/kg). The efficacy of 2,4-ds-PBN was assessed withthe tumor growth patterns and growth rates by using magnetic resonance(MR) imaging (T1/T2-weighted imaging) methods over the course of amonth.

FIGS. 1A-C show an example of the progressive development of a ratglioma at 10 (FIG. 1A), 15 (FIG. 1B) and 18 (FIG. 1C) days afterintracerebral implantation of C6 rat glioma cells. Histologicalevaluations were also done for the C6 rat glioma model. FIGS. 2-1(a)-2-2(c) show representative histological slides from regionsassociated with the glioma and comparative control tissue in thecontralateral side. Immunohistochemistry data for the von Willebrandfactor indicates endothelial cell proliferation is present in thenecrotic centers (FIG. 2-2 c), and small blood vessels are revealed inother parts of the glioma (FIG. 2-2 b), whereas there is no presence ofnew vasculature in the control side (FIG. 2-2 a), indicating thatangiogenesis associated with glioma formation is present.

FIG. 4 represents a series of MR images taken over a period of 25 daysfor an untreated control group and representative data (best-case,average effect, and worst-case) for 2,4-ds-PBN-treated rats (viadrinking water administration at a dose of 17 mg/kg/day). The 2,4-ds-PBNstudies indicated that a similar effect was observed as seen for PBN,where the C6 glioma tumor volumes were significantly decreased (>15-folddecrease; n=15 for 2,4-ds-PBN-treated rats, and n=5 for non-treatedrats) in 2,4-ds-PBN-treated rats (see FIG. 5). Rats treated with2,4-ds-PBN achieved >80% survival over the 40 day study period (n=15),compared to non-treated rats (n=5). No side effects were observed duringthe treatment period. Gross signs of adverse effects that were monitoredincluded weight loss, ruffling of fur, and behavioural and posturalchanges. No toxicity effects were detected (gross examination oftissues/organs during necropsies and histological assessment). FIG. 6illustrates one of the possible mechanisms of 2,4-ds-PBN, where in C6cells, it is found to be pro-apoptotic as concentration increases.

However, studies also showed that PBN significantly inhibited cellproliferation in a concentration dependent manner, but by contrast,2,4-ds-PBN and S-PBN showed little effect. The inventors then examinediNOS and VEGF protein expressions in the 2,4-ds-PBN treated C6 cells.2,4-ds-PBN (100 μM) treatment significantly lowered the iNOS expression(approximately 75% decrease) as compared with PBN (5% decrease at 100μM), and VEGF expression was reduced by 50% when cells were treated with5 mM 2,4-ds-PBN. These results suggest that tumor suppression by2,4-ds-PBN depends on decreased angiogenesis, decreased iNOS, as well asincreased apoptosis rather than cell growth inhibition. The effect ofeach nitrone compound in cells showed the same tendency as in the ratmodel, suggesting that the cellular model may replicate in vivo tumorregression mechanism in this model. From these studies, it can beconcluded that 2,4-ds-PBN significantly decreases tumor volumes as wellas increases subject survivability.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of treating a glioma in a subject comprising administeringto said subject a dose of 2,4-disulfonyl phenyl tert-butyl nitrone(2,4-ds-PBN) effective to inhibit the vascularization, growth or spreadof said glioma.
 2. The method of claim 1, wherein administration isthrough a route requiring subsequent passage of 2,4-ds-PBN across theblood brain barrier.
 3. The method of claim 2, wherein the route isenteral, intravenous, or intaarterial.
 4. The method of claim 1, whereinthe human subject has a recurrent or metastatic glioma.
 5. The method ofclaim 1, wherein the human subject has previously failed one or moreanti-glioma therapies.
 6. The method of claim 1, wherein the effectivedose is from about 5 to about 150 mg/kg body weight per day.
 7. Themethod of claim 2, wherein enteral administration is through dietarysupplementation of a food component.
 8. The method of claim 2, whereinthe enteral administration is in the form of a pill or a liquid.
 9. Themethod of claim 1, further comprising a secondary anti-glioma therapy.10. The method of claim 9, wherein the secondary anti-glioma therapy isradiation, surgery, or chemotherapy, such as lomustine, vincristine,matulane, PCV, BCNU, CCNU and/or DFMO.
 11. The method of claim 7,wherein the effective amount is from about 0.005 w/w % to about 0.1 w/w% of the diet being administered.
 12. The method of claim 1, whereinsaid glioma is an astrocytoma, an oligodendroglioma, or a glioblastomamultiforme.
 13. A method for inhibiting glioma development in a subjectcomprising (a) identifying a subject at risk of developing a glioma and(b) administering to said subject a dose of 2,4-disulfonyl phenyltert-butyl nitrone (2,4-ds-PBN) effective to inhibit the development ofsaid glioma.
 14. The method of claim 13, wherein administration isthrough a route requiring subsequent passage of 2,4-ds-PBN across theblood brain barrier.
 15. The method of claim 14, wherein the route isenteral, intravenous, or intaarterial.
 16. The method of claim 13,wherein the subject has a familial history of cancer or has been exposedto a carcinogenic environment.
 17. The method of claim 16, whereinspecific glioma risk factors include exposure to N-nitroso compounds orX-irradiation.
 18. The method of claim 13, wherein the effective dose isfrom about 5 to about 150 mg/kg body weight per day.
 19. The method ofclaim 15, wherein enteral administration is through dietarysupplementation of a food component.
 20. The method of claim 15, whereinthe enteral administration is in the form of a pill or a liquid.
 21. Themethod of claim 20, wherein the effective amount is from about 0.005 w/w% to about 0.1 w/w % of the diet being administered.
 22. The method ofclaim 13, wherein said glioma is an astrocytoma, an oligodendroglioma,or a glioblastoma multiforme.
 23. A method for inhibiting gliomarecurrence comprising administering to a subject previously having aglioma a dose of 2,4-disulfonyl phenyl tert-butyl nitrone (2,4-ds-PBN)effective to inhibit the development of said glioma.
 24. The method ofclaim 23, wherein administration is through a route requiring subsequentpassage of 2,4-ds-PBN across the blood brain barrier.
 25. The method ofclaim 24, wherein the route is enteral, intravenous, or intaarterial.26. The method of claim 23, wherein said glioma is an astrocytoma, anoligodendroglioma, or a glioblastoma multiforme.
 27. The method of claim23, wherein the effective dose is from about 5 to about 150 mg/kg bodyweight per day.
 28. The method of claim 23, further comprisingadministering a second agent that inhibits glioma occurrence.
 29. Themethod of claim 23, further comprising screening for glioma formation insaid subject.